Conventional and molecular diagnostics in onychomycosis-part2 : Molecular identification of causative dermatophytes by polymerase chain reaction and sequence analysis of the internal transcribed spacer region of ribosomal DNA

Abstract

Dermatophyte identification using traditional methods such as optics-based direct fluorescence microscopy and culture is nowadays supplemented by molecular biological methods. The validity of dermatophyte DNA detection with direct uniplex-polymerase chain reaction-enzyme immunoassay (PCR-EIA) in nail samples was proven by sequence analysis of the ribosomal internal transcribed spacer (ITS) region. A total of 108dermatophytes, isolated from patients with onychomycosis, were positive for Trichophyton rubrum (TR) and Trichophyton interdigitale (TI) in culture and/or uniplex-PCR-EIA. Conventional methods for dermatophyte identification were complemented by direct uniplex-PCR-EIA and sequence analysis of the ribosomal ITS region (18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA). Of 108 patients (average age62, median age 73), 56showed cultural growth with 31of them being identified as TR and23 as TI. There was high agreement with the sequence analysis. Surprisingly, the pathogen of asingle nail sample was identified as T.quinckeanum (formerly T.mentagrophytes sensu stricto), arare zoophilic dermatophyte in Germany. Asingle TI strain turned out to be amisidentified T.tonsurans based on the sequence analysis. In all, 34of the 52specimens lacking cultural growth were detected by PCR as TR, and 18specimens could be identified as TI. The results of dermatophyte identification of culture-negative nail samples were also in agreement with the results of sequence analysis. Molecular biological methods are well applicable, and they show high reliability for direct dermatophyte identification in nail samples without prior cultivation. Especially for nail samples without cultural growth, PCR-based dermatophyte identification was highly specific and sensitive.